| Objective: The aminoglucomannan(AGM) is a kind of semi-artificial product with cationic identity which is a common property of non-viral carrier for gene therapy. This cationic polysaccharide was characterized in this paper at the size of particles, the chromatography, molecular weight and cationic degree. The application of the AGM-pDNA complex was explored, and some conditions affecting the complex forming process were also studied. Its function for cell transfected was identified by AGM as a non-viral carrier transferring pEGFP-C1 gene or oligonucleotide into HEK293 cells. The safety and effect of AGM as DNA carrier were investigated in the cultured cells. We hope to develop a nevol gene carrier that can be used in vitro and in vivo. Methods: 1. Preparation of AGM: glucomannan(GM) solution was deposited by ethanol, then the precipitation was redissolved with deionic aqua. The AGM was prepared from 20mL GM solution which contained various proportion of ammonia, and the reaction of ammonification was catalyzed by ultrosonic. 2. The physico-chemical properties of AGM: (1) The molecular weight and purity of GM and AGM were analyzed by high performance liquid chromatography(HPLC). (2) The particle of AGM was observed under light microscopy. (3) The conductivity and TDS of the AGM were measured by conductivity meter. (4) The wavelength of maximum absorbance of ammonia, GM, AGM, and glucomannan contained ammonia were studied with UV spectrophotometer. 3.The DNA condensation effect of AGM: The cationic polysaccharide and pDNA complex was analyzed by electrophoresis according to the change of shape and mobility of bands. Some conditions (concentration, cationic degree and pH of AGM and so on) effecting the formation and stability of complex were investigated respectively. 4. The AGM cytotoxicity: The HEK293 cells were cultured in medium contained 1.25%~10%(v/v) AGM for 48h, then the OD value of each group was measured by MTT assay. The statistical compare was analyzed by t-test. 5. The transfection of AGM-pEGFP-C1 complex: The HEK293 cells was selected to perform the transfection effect of AGM.Five groups were designed: A: the preheating group; B: the post-heating group; C: the non-heating group; D: control group; E: the positive group. The complex prepared of 30μL, 20μL, 10μL, and 5μL AGM and 0.5μg pEGFP-C1 per well in the group A,B, and C. The cultured cells were observed with fluorescent microscopy 24h later and the GFP was counted. 6. The transfection of AGM-oligonucleotide complex: The complex prepared of AGM and oligonucleotide labeled by Rhodamine was added to HEK293 cells. The red spots were observed within cells by fluorescent microscope 5-24h later. Results: 1. The AGM properties: (1) AGM could be aggregating into various particles responsible to the degree of ammonification. (2) The conductivity and TDS of AGM is increasing to 30 folds than those of glucomannan. (3) The purity of GM and AGM were 100% and 93.5%. The molecular weight of them are about 80000 Dolton. (4) Its absorption peak (λmax)at 209nm is tailing, and the wavelength was just approached that of ammonia (λmax=207nm); The form of peak was same to the GM (λmax=192nm). It has the characteristics of both ammonia and GM.2. The condensation of AGM and pDNA: Compared with the pDNA, the AGM-pDNA complex appeared the change of shape, reducing mobility of bands, even retarded in the gel well. The complex is increasing with the concentration or amonification degree of AGM.The optimal pH is 6-7. When the concentration of NaCl was decreased in the solution, the complex formation was increased, and the complex was more stable than those in higher. When AGM was pretreated at 80℃ for one or two hours, It was weak or incapable of being condensation with pDNA.3. The cytotoxicity evaluation of the AGM: The OD490 of HEK293 cells is a little higher in the cells culture with 2.5% to 10% AGM than that in the control group(p<0.01). 4. AGM transferred the pDNA into the cells: The means of green spots in each group were investigated... |
PDF Full Text Request